Intestinal bacteria carry antimicrobial resistance (AMR) genes in mobile genetic elements which have the potential to spread to bacteria in other animal hosts including humans. In fecal matter, Escherichia coli can continue to multiply for 48 hr after being excreted, and in certain environments, E. coli survive long periods of time. It is unclear the extent to which AMR in E. coli changes in the environment outside of its host. In this study, we analyzed changes in the population structure, plasmid content, and AMR patterns of 30 E. coli isolates isolated from 6 chickens (cloacal swabs), and 30 E. coli isolates from fecal samples (from the same 6 chickens) after 24 hr of incubation. Clonality of isolates was screened using the fumC gene sequence and confirmed in a subset of isolates (n = 14) by multi-locus sequence typing. Major shifts in the population structure (i.e., sequence types) and antibiotic resistance patterns were observed among the numerically dominant E. coli isolates after 24 hr. Four E. coli clones isolated from the cloaca swabs and the corresponding fecal samples (after 24 hr incubation) showed different antibiotic resistance patterns. Our study reveals that fecal matter in the environment is an intermediate habitat where rapid and striking changes occur in E. coli populations and antibiotic resistance patterns.